Conditional Inhibition of Eip75B Eliminates the Effects of Mating and Mifepristone on Lifespan in Female Drosophila.

Mating in female Drosophila melanogaster causes midgut hypertrophy and reduced lifespan, and these effects are blocked by the drug mifepristone. Eip75B is a transcription factor previously reported to have pleiotropic effects on Drosophila lifespan. Because Eip75B null mutations are lethal, conditional systems and/or partial knock-down are needed to study Eip75B effects in adults. Previous studies showed that Eip75B is required for adult midgut cell proliferation in response to mating. To test the possible role of Eip75B in mediating the lifespan effects of mating and mifepristone, a tripartite FLP-recombinase-based conditional system was employed that provides controls for genetic background. Expression of a Hsp70-FLP transgene was induced in third instar larvae by a brief heat pulse. The FLP recombinase catalyzed the recombination and activation of an Actin5C-GAL4 transgene. The GAL4 transcription factor in turn activated expression of a UAS-Eip75B-RNAi transgene. Inhibition of Eip75B activity was confirmed by loss of midgut hypertrophy upon mating, and the lifespan effects of both mating and mifepristone were eliminated. In addition, the negative effects of mifepristone on egg production were eliminated. The data indicate that Eip75B mediates the effects of mating and mifepristone on female midgut hypertrophy, egg production, and lifespan.

Dhr96[1] mutation and maternal tudor[1] mutation increase life span and reduce the beneficial effects of mifepristone in mated female Drosophila.

Mating and receipt of male Sex Peptide hormone cause increased egg laying, increased midgut size and decreased life span in female Drosophila. Feeding mated females with the synthetic steroid mifepristone decreases egg production, reduces midgut size, and increases life span. Here, several gene mutations were assayed to investigate possible mechanisms for mifepristone action. Drosophila Dhr96 is a hormone receptor, and a key positive regulator of midgut lipid uptake and metabolism. Dhr96[1] null mutation increased female life span, and reduced the effects of mifepristone on life span, suggesting that Dhr96[1] mutation and mifepristone may act in part through the same mechanism. Consistent with this idea, lipidomics analysis revealed that mating increases whole-body levels of triglycerides and fatty-acids in triglycerides, and these changes are reversed by mifepristone. Maternal tudor[1] mutation results in females that lack the germ-line and produce no eggs. Maternal tudor[1] mutation increased mated female life span, and reduced but did not eliminate the effects of mating and mifepristone on life span. This indicates that decreased egg production may be related to the life span benefits of mifepristone, but is not essential. Mifepristone increases life span in w[1118] mutant mated females, but did not increase life span in w[1118] mutant virgin females. Mifepristone decreased egg production in w[1118] mutant virgin females, indicating that decreased egg production is not sufficient for mifepristone to increase life span. Mifepristone increases life span in virgin females of some, but not all, white[+] and mini-white[+] strains. Backcrossing of mini-white[+] transgenes into the w[1118] background was not sufficient to confer a life span response to mifepristone in virgin females. Taken together, the data support the hypothesis that mechanisms for mifepristone life span increase involve reduced lipid uptake and/or metabolism, and suggest that mifepristone may increase life span in mated females and virgin females through partly different mechanisms.

A screen of small molecule and genetic modulators of life span in female Drosophila identifies etomoxir, RH5849 and unanticipated temperature effects.

Mifepristone increases life span in female Drosophila melanogaster, and its molecular target(s) remain unclear. Here small molecule and genetic interventions were tested for ability to mimic mifepristone, or to decrease life span in a way that can be rescued by mifepristone. Etomoxir inhibits lipid metabolism, and significantly increased life span in virgin and mated females, but not males, at 50 µM concentration. Pioglitazone is reported to activate both mammalian PPARγ and its Drosophila homolog Eip75B. Pioglitazone produced minor and inconsistent benefits for female Drosophila life span, and only at the lowest concentrations tested. Ecdysone is a Drosophila steroid hormone reported to regulate responses to mating, and RH5849 is a potent mimic of ecdysone. RH5849 reduced virgin female life span, and this was partly rescued by mifepristone. Mifepristone did not compete with RH5849 for activation of an ecdysone receptor (EcR)-responsive transgenic reporter, indicating that the relevant target for mifepristone is not EcR. The conditional GAL4/GAL80ts system was used in attempt to test the effect of an Eip75B RNAi construct on female life span. However, the 29°C temperature used for induction reduced or eliminated mating-induced midgut hypertrophy, the negative life span effects of mating, and the positive life span effects of mifepristone. Even when applied after mating was complete, a shift to 29°C temperature reduced mating-induced midgut hypertrophy by half, and the life span effects of mating by 4.8-fold. Taken together, these results identify promising small molecules for further analysis, and inform the design of experiments involving the GAL4/GAL80ts system.

Mifepristone Increases Life Span in Female Drosophila Without Detectable Antibacterial Activity.

Mifepristone dramatically increases the life span of mated female Drosophila while reducing the expression of innate immune response genes. Previous results indicated that mifepristone also reduced the load of aero-tolerant bacteria in mated females. Experiments were conducted to further investigate the possible role of bacteria in mifepristone life span effects. Life span was assayed in flies grown from sterilized eggs on autoclaved media and in normally cultured controls in two independent assays. Sterilization increased mated female life span (+8.3% and +57%, respectively), and the effect of mifepristone was additive (+53% and +93%, respectively). High-throughput sequencing of 16S sequences revealed that sterilization reduced the abundance of multiple species and the classes Bacteroidia, Bacilli, Actinobacteria, and Cytophagia. By contrast, mifepristone caused no decreases and instead increased the abundance of three species. Five aero-tolerant bacterial species were cultured from extracts of mated female flies, including both Gram-positive and Gram-negative species (Acetobacter sicerae, Enterococcus faecalis, Lactobacillus plantarum, Serratia rubidea, and Paenibacillus glucanolyticus). There was no detectable effect of mifepristone on the growth of these bacteria in vitro, indicating that mifepristone does not have a direct antibiotic effect. To test if antibiotics could mimic the effects of mifepristone in vivo, mated female flies were treated throughout adult life span with high concentrations of the individual antibiotics doxycycline, ampicillin, kanamycin, and streptomycin, in replicate experiments. No significant effect on life span was observed for ampicillin, kanamycin, or streptomycin, and an inconsistent benefit was observed for doxycycline. Finally, supplementation of media with Enterococcus faecalis did not alter adult female life span in the presence or absence of mifepristone. Taken together, the results indicate the life span benefits of mifepristone are not due to an antibiotic effect.

Mifepristone Increases Life Span of Virgin Female Drosophila on Regular and High-fat Diet Without Reducing Food Intake.

Background: The synthetic steroid mifepristone is reported to have anti-obesity and anti-diabetic effects in mammals on normal and high-fat diets (HFD). We previously reported that mifepristone blocks the negative effect on life span caused by mating in female Drosophila melanogaster. Methods: Here we asked if mifepristone could protect virgin females from the life span-shortening effect of HFD. Mifepristone was assayed for effects on life span in virgin females, in repeated assays, on regular media and on media supplemented with coconut oil (HFD). The excrement quantification (EX-Q) assay was used to measure food intake of the flies after 12 days mifepristone treatment. In addition, experiments were conducted to compare the effects of mifepristone in virgin and mated females, and to identify candidate mifepristone targets and mechanisms. Results: Mifepristone increased life span of virgin females on regular media, as well as on media supplemented with either 2.5 or 5% coconut oil. Food intake was not reduced in any assay, and was significantly increased by mifepristone in half of the assays. To ask if mifepristone might rescue virgin females from all life span-shortening stresses, the oxidative stressor paraquat was tested, and mifepristone produced little to no rescue. Analysis of extant metabolomics and transcriptomics data suggested similarities between effects of mifepristone in virgin and mated females, including reduced tryptophan breakdown and similarities to dietary restriction. Bioinformatics analysis identified candidate mifepristone targets, including transcription factors Paired and Extra-extra. In addition to shortening life span, mating also causes midgut hypertrophy and activation of the lipid metabolism regulatory factor SREBP. Mifepristone blocked the increase in midgut size caused by mating, but did not detectably affect midgut size in virgins. Finally, mating increased activity of a SREBP reporter in abdominal tissues, as expected, but reporter activity was not detectably reduced by mifepristone in either mated or virgin females. Conclusion: Mifepristone increases life span of virgin females on regular and HFD without reducing food intake. Metabolomics and transcriptomics analyses suggest some similar effects of mifepristone between virgin and mated females, however reduced midgut size was observed only in mated females. The results are discussed regarding possible mifepristone mechanisms and targets.

Metabolic Signatures of Life Span Regulated by Mating, Sex Peptide, and Mifepristone/RU486 in Female Drosophila melanogaster.

Mating and transfer of male sex peptide (SP), or transgenic expression of SP, causes inflammation and decreased life span in female Drosophila. Mifepristone rescues these effects, yielding dramatic increases in life span. Here targeted metabolomics data were integrated with further analysis of extant transcriptomic data. Each of 7 genes positively correlated with life span were expressed in the brain or eye and involved regulation of gene expression and signaling. Genes negatively correlated with life span were preferentially expressed in midgut and involved protein degradation, amino acid metabolism, and immune response. Across all conditions, life span was positively correlated with muscle breakdown product 1/3-methylhistidine and purine breakdown product urate, and negatively correlated with tryptophan breakdown product kynurenic acid, suggesting a SP-induced shift from somatic maintenance/turnover pathways to the costly production of energy and lipids from dietary amino acids. Some limited overlap was observed between genes regulated by mifepristone and genes known to be regulated by ecdysone; however, mifepristone was unable to compete with ecdysone for activation of an ecdysone-responsive transgenic reporter. In contrast, genes regulated by mifepristone were highly enriched for genes regulated by juvenile hormone (JH), and mifepristone rescued the negative effect of JH analog methoprene on life span in adult virgin females. The data indicate that mifepristone increases life span and decreases inflammation in mated females by antagonizing JH signaling downstream of male SP. Finally, mifepristone increased life span of mated, but not unmated, Caenorhabditis elegans, in 2 of 3 trials, suggesting possible evolutionary conservation of mifepristone mechanisms.

Analysis of Drosophila melanogaster Lifespan.

The laboratory fruit fly Drosophila melanogaster is one of the leading models for the study of aging. Whereas several behavioral and physiological biomarkers of aging have been identified for Drosophila, lifespan remains the most robust measure of aging rate. Aging and lifespan can be modulated by genetic alterations, as well as by drugs and dietary components, to reveal basic and conserved mechanisms of aging. Here methods are presented for Drosophila lifespan assay, including media preparation, supplementation of media with various drugs, culturing of the flies, passaging flies and recording deaths, and the analysis of lifespan data.

Mifepristone/RU486 acts in Drosophila melanogaster females to counteract the life span-shortening and pro-inflammatory effects of male Sex Peptide.

Males with null mutation of Sex Peptide (SP) gene were compared to wild-type males for the ability to cause physiological changes in females that could be reversed by mifepristone. Males from wild-type strains decreased median female life span by average -51%. Feeding mifepristone increased life span of these females by average +106%. In contrast, SP-null males did not decrease female life span, and mifepristone increased median life span of these females by average +14%, which was equivalent to the effect of mifepristone in virgin females (average +16%). Expression of innate immune response transgenic reporter (Drosocin-GFP) was increased in females mated to wild-type males, and this expression was reduced by mifepristone. In contrast, SP-null males did not increase Drosocin-GFP reporter expression in the female. Similarly, mating increased endogenous microbial load, and this effect was reduced or absent in females fed mifepristone and in females mated to SP-null males; no loss of intestinal barrier integrity was detected using dye-leakage assay. Reduction of microbial load by treating adult flies with doxycycline reduced the effects of both mating and mifepristone on life span. Finally, mifepristone blocked the negative effect on life span caused by transgenic expression of SP in virgin females. The data support the conclusion that the majority of the life span-shortening, immune-suppressive and pro-inflammatory effects of mating are due to male SP, and demonstrate that mifepristone acts in females to counteract these effects of male SP.

Multiple Metazoan Life-span Interventions Exhibit a Sex-specific Strehler-Mildvan Inverse Relationship Between Initial Mortality Rate and Age-dependent Mortality Rate Acceleration.

The Gompertz equation describes survival in terms of initial mortality rate (parameter a), indicative of health, and age-dependent acceleration in mortality rate (parameter b), indicative of aging. Gompertz parameters were analyzed for several published studies. In Drosophila females, mating increases egg production and decreases median life span, consistent with a trade-off between reproduction and longevity. Mating increased parameter a, causing decreased median life span, whereas time parameter b was decreased. The inverse correlation between parameters indicates the Strehler-Mildvan (S-M) relationship, where loss of low-vitality individuals yields a cohort with slower age-dependent mortality acceleration. The steroid hormone antagonist mifepristone/RU486 reversed these effects. Mating and mifepristone showed robust S-M relationships across genotypes, and dietary restriction showed robust S-M relationship across diets. Because nutrient optima differed between females and males, the same manipulation caused opposite effects on mortality rates in females versus males across a range of nutrient concentrations. Similarly, p53 mutation in Drosophila and mTOR mutation in mice caused increased median life span associated with opposite direction changes in mortality rate parameters in females versus males. The data demonstrate that dietary and genetic interventions have sex-specific and sometimes sexually opposite effects on mortality rates consistent with sexual antagonistic pleiotropy.

The progesterone antagonist mifepristone/RU486 blocks the negative effect on life span caused by mating in female Drosophila.

Mating causes decreased life span in female Drosophila. Here we report that mifepristone blocked this effect, yielding life span increases up to +68%. Drug was fed to females after mating, in the absence of males, demonstrating function in females. Mifepristone did not increase life span of virgin females or males. Mifepristone reduced progeny production but did not reduce food intake. High-throughput RNA sequencing was used to identify genes up-regulated or down-regulated upon mating, and where the change was reduced by mifepristone. Five candidate positive regulators of life span were identified, including dosage compensation regulator Unr and three X-linked genes: multi sex combs (PcG gene), Dopamine 2-like receptor and CG14215. The 37 candidate negative genes included neuropeptide CNMamide and several involved in protein mobilization and immune response. The results inform the interpretation of experiments involving mifepristone, and implicate steroid hormone signaling in regulating the trade-off between reproduction and life span.

Variegated expression of Hsp22 transgenic reporters indicates cell-specific patterns of aging in Drosophila oenocytes.

The cytoplasmic chaperone gene Hsp70 and the mitochondrial chaperone gene Hsp22 are upregulated during normal aging in Drosophila in tissue-general patterns. In addition, Hsp22 reporters are dramatically upregulated during aging in a subset of the oenocytes (liver-like cells). Hsp22 reporter expression varied dramatically between individual oenocytes and between groups of oenocytes located in adjacent body segments, and was negatively correlated with accumulation of age pigment, indicating cell-specific and cell-lineage-specific patterns of oenocyte aging. Conditional transgenic systems were used to express 88 transgenes to search for trans-regulators of the Hsp70 and Hsp22 reporters during aging. The wingless gene increased tissue-general upregulation of both Hsp70 and Hsp22 reporters. In contrast, the mitochondrial genes MnSOD and Hsp22 increased expression of Hsp22 reporters in the oenocytes and decreased accumulation of age pigment in these cells. The data suggest that cell-specific and cell lineage-specific patterns of mitochondrial malfunction contribute to oenocyte aging.

Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster.

Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging.

Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster.

Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA with a two-base deletion. If the deletion occurs in a coding region, translation will result in production of misframed proteins. During mammalian aging, misframed versions of human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in the aggregates characteristic of neurodegenerative diseases, suggesting dysfunctional degradation or clearance. Here cDNA clones encoding wild-type hUbb and the frame-shifted version hUbb(+1) were expressed in transgenic Drosophila using the doxycycline-regulated system. Misframed proteins were abundantly produced, both from the transgenes and from endogenous Drosophila ubiquitin-encoding genes, and their abundance increased during aging in whole-fly extracts. Over-expression of wild-type hUbb, but not hUbb(+1), was toxic during fly development. In contrast, when over-expressed specifically in adult flies, hUbb(+1) caused small decreases in life span, whereas hUbb was associated with small increases, preferentially in males. The data suggest that MM occurs in Drosophila and that the resultant misframed proteins accumulate with age. MM of the ubiquitin gene can produce alternative ubiquitin gene products with different and sometimes opposing phenotypic effects.

Identifying sexual differentiation genes that affect Drosophila life span.

BACKGROUND: Sexual differentiation often has significant effects on life span and aging phenotypes. For example, males and females of several species have different life spans, and genetic and environmental manipulations that affect life span often have different magnitude of effect in males versus females. Moreover, the presence of a differentiated germ-line has been shown to affect life span in several species, including Drosophila and C. elegans. METHODS: Experiments were conducted to determine how alterations in sexual differentiation gene activity might affect the life span of Drosophila melanogaster. Drosophila females heterozygous for the tudor[1] mutation produce normal offspring, while their homozygous sisters produce offspring that lack a germ line. To identify additional sexual differentiation genes that might affect life span, the conditional transgenic system Geneswitch was employed, whereby feeding adult flies or developing larvae the drug RU486 causes the over-expression of selected UAS-transgenes. RESULTS: In this study germ-line ablation caused by the maternal tudor[1] mutation was examined in a long-lived genetic background, and was found to increase life span in males but not in females, consistent with previous reports. Fitting the data to a Gompertz-Makeham model indicated that the maternal tudor[1] mutation increases the life span of male progeny by decreasing age-independent mortality. The Geneswitch system was used to screen through several UAS-type and EP-type P element mutations in genes that regulate sexual differentiation, to determine if additional sex-specific effects on life span would be obtained. Conditional over-expression of transformer female isoform (traF) during development produced male adults with inhibited sexual differentiation, however this caused no significant change in life span. Over-expression of doublesex female isoform (dsxF) during development was lethal to males, and produced a limited number of female escapers, whereas over-expression of dsxF specifically in adults greatly reduced both male and female life span. Similarly, over-expression of fruitless male isoform A (fru-MA) during development was lethal to both males and females, whereas over-expression of fru-MA in adults greatly reduced both male and female life span. CONCLUSION: Manipulation of sexual differentiation gene expression specifically in the adult, after morphological sexual differentiation is complete, was still able to affect life span. In addition, by manipulating gene expression during development, it was possible to significantly alter morphological sexual differentiation without a significant effect on adult life span. The data demonstrate that manipulation of sexual differentiation pathway genes either during development or in adults can affect adult life span.

Drosophila melanogaster p53 has developmental stage-specific and sex-specific effects on adult life span indicative of sexual antagonistic pleiotropy.

Truncated and mutant forms ofp53 affect life span in Drosophila, nematodes and mice, however the role of wild-type p53 in aging remains unclear. Here conditional over-expression of both wild-type and mutant p53 transgenes indicated that, in adult flies, p53 limits life span in females but favors life span in males. In contrast, during larval development, moderate over-expression of p53 produced both male and female adults with increased life span. Mutations of the endogenous p53 gene also had sex-specific effects on life span under control and stress conditions: null mutation of p53 increased life span in females, and had smaller, more variable effects in males. These developmental stage-specific and sex-specific effects of p53 on adult life span are consistent with a sexual antagonistic pleiotropy model.

Conditional switches for extracellular matrix patterning in Drosophila melanogaster.

An F(1) mutagenesis strategy was developed to identify conditional mutations affecting extracellular matrix (ECM) patterning. Tubulogenesis requires coordinated movement of epithelial cells and deposition of a multilayered ECM. In the Drosophila ovary, an epithelium of follicle cells creates the eggshells, including the paired tubular dorsal appendages (DAs) that act as breathing tubes for the embryo. A P-element mutagenesis strategy allowed for conditional overexpression of hundreds of genes in follicle cells. Conditional phenotypes were scored at the level of individual mutant (F(1)) female flies. ECM pattern regulators were readily identified including MAPK signaling gene ets domain lacking (fused DAs), Wnt pathway genes frizzled 3 and osa (long DAs), Hh pathway gene debra (branched DAs), and transcription factor genes sima/HIF-1alpha, ush, lilli, Tfb1, broad, and foxo. In moving cells the [Ca(2+)]/calcineurin pathway can regulate adhesion to ECM while adherens junctions link cells together. Accordingly, thin eggshell and DA phenotypes were identified for the calcineurin regulator calreticulin and the adherens junction component arc. Finally a tubulogenesis defect phenotype was identified for the gene pterodactyl, homologous to the mammalian serine/threonine receptor-associated protein (STRAP) that integrates the TGF-beta and PI3K/AKT signaling pathways. Because phenotypes can be scored in each mutant fly before and after gene induction, this F(1) conditional mutagenesis strategy should allow for increased scale in screens for mutations affecting repeated (reiterated) events in adult animals, including gametogenesis, movement, behavior, and learning.

Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes.

BACKGROUND: Several interventions increase lifespan in model organisms, including reduced insulin/insulin-like growth factor-like signaling (IIS), FOXO transcription factor activation, dietary restriction, and superoxide dismutase (SOD) over-expression. One question is whether these manipulations function through different mechanisms, or whether they intersect on common processes affecting aging. RESULTS: A doxycycline-regulated system was used to over-express manganese-SOD (MnSOD) in adult Drosophila, yielding increases in mean and maximal lifespan of 20%. Increased lifespan resulted from lowered initial mortality rate and required MnSOD over-expression in the adult. Transcriptional profiling indicated that the expression of specific genes was altered by MnSOD in a manner opposite to their pattern during normal aging, revealing a set of candidate biomarkers of aging enriched for carbohydrate metabolism and electron transport genes and suggesting a true delay in physiological aging, rather than a novel phenotype. Strikingly, cross-dataset comparisons indicated that the pattern of gene expression caused by MnSOD was similar to that observed in long-lived Caenorhabditis elegans insulin-like signaling mutants and to the xenobiotic stress response, thus exposing potential conserved longevity promoting genes and implicating detoxification in Drosophila longevity. CONCLUSION: The data suggest that MnSOD up-regulation and a retrograde signal of reactive oxygen species from the mitochondria normally function as an intermediate step in the extension of lifespan caused by reduced insulin-like signaling in various species. The results implicate a species-conserved net of coordinated genes that affect the rate of senescence by modulating energetic efficiency, purine biosynthesis, apoptotic pathways, endocrine signals, and the detoxification and excretion of metabolites.

Alteration of Drosophila life span using conditional, tissue-specific expression of transgenes triggered by doxycyline or RU486/Mifepristone.

The conditional systems Tet-on and Geneswitch were compared and optimized for the tissue-specific expression of transgenes and manipulation of life span in adult Drosophila. Two versions of Tet-on system reverse-tetracycline-Trans-Activator (rtTA) were compared: the original rtTA, and rtTAM2-alt containing mutations designed to optimize regulation and expression. The rtTAM2-alt version gave less leaky expression of target constructs in the absence of doxycyline, however the absolute level of expression that could be achieved was less than that produced by rtTA, in contrast to a previous report. Existing UAS-rtTAM2-alt insertions were re-balanced, and combined with several tissue-general and tissue-specific GAL4 driver lines to yield tissue-specific, doxycyline-inducible transgene expression over three orders of magnitude. The Geneswitch (GS) system also had low background, but the absolute level of expression was low relative to Tet-on. Consequently, actin5C-GS multi-insert chromosomes were generated and higher-level expression was achieved without increased background. Moderate level over-expression of MnSOD has beneficial effects on life span. Here high-level over-expression of MnSOD was found to have toxic effects. In contrast, motor-neuron-specific over-expression of MnSOD had no detectable effect on life span. The results suggest that motor-neuron tissue is not the essential tissue for either MnSOD induced longevity or toxicity in adult males.

Superoxide dismutase evolution and life span regulation.

Superoxide is among the most abundant reactive oxygen species (ROS) produced by the mitochondria, and is involved in cellular signaling pathways. Superoxide and other ROS can damage cellular macromolecules and levels of oxidative damage products are positively correlated with aging. Superoxide dismutase (SOD) enzymes catalyze the breakdown of superoxide into hydrogen peroxide and water and are therefore central regulators of ROS levels. Genetic and transgenic manipulation of SOD activities in model systems such as S. cereviseae, mouse and Drosophila are consistent with a central role for SOD enzymes in regulating oxidative stress resistance. Over-expression of SOD in S. cereviseae and Drosophila can reduce oxidative damage and extend life span, but the mechanism(s) are not yet clear. A phylogenetic analysis of publicly available SOD protein sequences suggests several additional conserved gene families. For example, in addition to the well-characterized soluble Cu/Zn enzyme (Sod) and mitochondrial manganese-containing form (Sod2), Drosophila melanogaster is found to contain a putative copper chaperone (CCS), an extracellular Cu/Zn enzyme (Sod3), and an extracellular protein distantly related to the Cu/Zn forms (Sodq). C. elegans and blue crab are unusual in having two Mn-containing SODs, and A. gambiae contains an unusual internally repeated SOD. The most parsimonius conclusion from the analysis of the extracellular SODs is that they evolved independently multiple times by addition of a signal peptide to cytoplasmic SOD.